Lung cancer is the leading cause of cancer-related mortality in both men and women. The prevalence of lung cancer is second only to that of prostate cancer in men and breast cancer in women. Lung cancer recently surpassed heart disease as the leading cause of smoking-related mortality. In addition, most patients who develop lung cancer smoke and have smoking-related damage to the heart and lungs, making aggressive surgical or multimodality therapies less viable options. Most lung carcinomas are diagnosed at an advanced stage, conferring a poor prognosis.
Non-small cell lung cancer (NSCLC) accounts for approximately 75% of all lung cancers. NSCLC is a heterogeneous aggregate of histologies. The most common histologies are epidermoid or squamous carcinoma, adenocarcinoma, and large cell carcinoma.
Several studies have attempted to identify clinical, laboratory, and molecular markers that may help clinicians and researchers distinguish subgroups of NSCLC patients. Along these lines, various studies have shown that epidermal growth factor receptor (EGFR) is over-expressed in 40 to 80 percent of non-small cell lung cancers and many other epithelial cancers.
Aberrant epidermal growth factor receptor (EGFR) signalling is critical for limiting sensitivity to anticancer agents and ligand-independent tyrosine kinase activation of EGFR is often caused by EGFR mutations in the extracellular domain, which has been observed in various tumour types, such as glioblastoma multiforme. EGFR signalling is triggered by the binding of growth factors, such as epidermal growth factor (EGF). Autophosphorylation and transphosphorylation of the receptors through their tyrosine kinase domains leads to the recruitment of downstream effectors and the activation of proliferative and cell-survival signals.
Two mutations account for approximately 90% of EGFR mutations reported to date in lung adenocarcinomas. In Caucasian population, the most common mutation type, seen in around 65% of cases with EGFR mutations, is a short in-frame deletion of 9, 12, 15, 18, or 24 nucleotides in exon 19. The second most common mutation, seen in about 35% of cases with EGFR mutations, is a point mutation (CTG to CGG) in exon 21 at nucleotide 2573, that results in substitution of leucine by arginine at codon 858 (L858R) adjacent to the DFG motif in the carboxy-terminal lobe in the activation loop of the kinase.
These EGFR mutations are bona fide somatic mutations in NSCLC and have not been identified in other primary tumour types. Further, EGFR mutations are a strong determinant of tumor response to gefitinib in non-small cell lung cancer (NSCLC). Other much less common mutations have been described in exons 18, 20, and 21.
So far, screening for these mutations has been based on direct sequencing or single-strand conformation polymorphism analysis. Nucleic acid amplification methods (for example, the polymerase chain reaction) allow the detection of small numbers of mutant molecules among a background of normal ones. While alternative means of detecting small numbers of tumor cells (such as flow cytometry) have generally been limited to hematological malignancies, nucleic acid amplification assays have proven both sensitive and specific in identifying malignant cells and for predicting prognosis following chemotherapy.
Various nucleic acid amplification strategies for detecting small numbers of mutant molecules in solid tumor tissue have been developed. For example, one sensitive and specific method identifies mutant ras oncogene DNA on the basis of failure to cleave a restriction site at the crucial 12th codon (Kahn et al. Rapid and sensitive nonradioactive detection of mutant K-ras genes via ‘enriched’ PCR amplification. Oncogene. 1991 June; 6(6):1079-83). Similar protocols can be applied to detect any mutated region of DNA in a neoplasm, allowing detection of other oncogene DNA or tumor-associated DNA. Since mutated DNA can be detected not only in the primary cancer but in both precursor lesions and metastatic sites, nucleic acid amplification assays provide a means of detecting and monitoring cancer both early and late in the course of disease.
Other studies have used nucleic acid amplification assays to analyze the peripheral blood of patients with cancer in order to detect intracellular DNA extracted from circulating cancer cells in patients. However, it must be emphasized that these studies attempt to use nucleic acid-based amplification assays to detect extracted intracellular DNA within circulating cancer cells. The assay is performed on the cellular fraction of the blood from patients having cancer using the cell pellet or cells within whole blood, and the serum or plasma fraction is conventionally ignored or discarded prior to analysis. Since such an approach requires the presence of metastatic circulating cancer cells (for non-hematologic tumors), it is of limited clinical use in patients with early cancers, and it is not useful in the detection of non-hematologic non-invasive neoplasms or pre-malignant states.
It is known in the prior art that small but significant amounts of normal DNA circulate in the blood of healthy people and this amount has been found to increase in cancer states. The prior art contains disclosure that mutant oncogene DNA could be detected in peripheral blood plasma or serum of cancer patients. However, these reports have also been generally limited to patients with advanced cancer or known neoplastic or proliferative disease. Some authors (Kimura et al., 2006. EGFR Mutation of Tumor and Serum in Gefitinib-Treated Patients with Chemotherapy-Naive Non-small Cell Lung Cancer) have described that mutations at EGFR gene can be detected in serum samples from patients suffering from NSCLC. Said document describes detection of such mutations by means of PCR and sequencing using primers flanking said mutations.